This application claims priority to Swedish Application No. SE 0101702-9 filed May 15, 2001 and United States Provisional Application No. 60/302,790 filed Jul. 3, 2001, each of which are hereby incorporated by reference.
This invention relates to novel compounds having therapeutic properties in themselves, and being capable of potentiating the efficacy of other therapeutically active compounds, for example cytotoxic compounds used in the treatment of cancer. The novel compounds have been shown to possess a cell growth inhibiting property, and in addition to this, also to increase the pharmacological activity of a conventional paclitaxel formulation and to make it possible to manufacture a new formulation of paclitaxel, exhibiting improved solubility and therapeutic efficacy.
While the term xe2x80x9cchemotherapyxe2x80x9d originally had a very broad meaning, encompassing the treatment of various diseases with chemical agents, it has today a more specific meaning. In modern language, the term xe2x80x9cchemotherapyxe2x80x9d usually refers to the use of chemical agents to destroy cancer cells. Among the chemical agents currently used as anticancer drugs, most function by impairing the ability of the cancer cells to replicate by interfering with DNA and RNA activities associated with cell division.
Paclitaxel is a diterpenoid compound {(2R,3S)-3-Benzamido-3-fenyl-2-hydroxy propionic acid-[(2aR, 4S, 4aS, 6R, 9S, 11S, 12S, 12aR, 12bS)-6,12b-diacetoxy-12-benzoyloxy-2a, 3, 4, 4a, 5, 6, 9, 10, 11, 12, 12a, 12b-dodecahydro-4,11-dihydroxy-4a, 8, 13, 13-tetramethyl-7,11-methano-5-oxo-1H-cyclodeca[3,4]benz[1,2-b]oxet-9-yl]ester, the active ingredient in Taxol(copyright), Bristol-Myers Squibb} originally isolated from the western yew, a cone-bearing evergreen tree of the genus Taxus. Paclitaxel is one example of an important chemotherapeutic agent or anticancer drug currently in use. It has a wide spectrum of activity against solid tumours: primarily breast cancer, ovarian, colon and non-small cell lung carcinomas. It binds to the xcex2-subunit of tubulin, resulting in the formation of stable non-functional microtubule bundles and thus interfering with mitosis. The drug can also induce apoptosis and has anti-angiogenic properties.
Paclitaxel is highly protein-bound, has large volumes of distribution, but poor penetration into the central nervous system. This compound is primarily eliminated from the body via hepatic metabolism, and its use is therefore generally precluded in severe hepatic dysfunction.
In recent years, considerable emphasis has been given to the development of new formulations of paclitaxel that are suitable for intravenous administration, in order to address the solubility and toxicity issues associated with this particular drug. Examples include dispersed systems such as emulsions, liposomes, mixed micelles prepared by co-precipitation using bile salts and phospholipids (Alkan-Onyuksel H, et al. Pharm. Res. vol 2. pp. 206-212, 1994), cyclodextrins, and microspheres. Water-soluble prodrugs such as polyethylene glycol- and polyglutamate-paclitaxel with promising antitumor activity have also been developed.
The commercially available product, Taxol(copyright) (a paclitaxel concentrate for preparation of solutions for infusion, marketed by Bristol-Myers Squibb Co., New York, N.Y., USA), is currently formulated in a vehicle containing a mixture of polyoxyethylated castor oil (Cremophor(copyright) EL) and ethanol, in the approximate proportions 1:1 (v/v). Cremophor(copyright) EL, which is a commonly used surfactant for lipophilic compounds, has however been associated with adverse side-effects, such as bronchospasms, hypotension, and other manifestations of hypersensitivity particularly following rapid administration. Therefor, long infusion times, high dilution of the ethanol:Cremophor(copyright) EL solution, and pre-medication (e.g. using corticosteroids, antihistamine, and H2-blockers) are actions resorted to in order to reduce these adverse effects.
Furthermore, the commercially available formulation is associated with a number of difficult technical issues such as stability, including the possibility of drug precipitation upon dilution, filtering requirements and restrictions regarding the use of PVC-containing vessels and administration sets. It is thus apparent that there is a need for a new formulation of paclitaxel that is efficacious and less toxic than the commercial product and which formulation can alleviate the side-effects and set aside the problems currently associated with preparation and administration of this drug.
Further, the small difference between the therapeutic and the toxic concentration severely limits the clinical usefulness of paclitaxel. The therapeutic efficacy could be improved by delivering the drug with an appropriate microcarrier system, which is able to change temporal and spatial biodistribution of the drug. This approach has been suggested for the highly toxic and poorly soluble amphotericin B, which has been successfully incorporated into disk-like micelles of cholersteryl sulphate (Lasic D. D. Nature. Vol. 355, 16 Jan., pp. 279-280, 1992).
In later years, great deal of effort has been given to the development of polymeric micellar paclitaxel formulations using amphiphilic diblock copolymers (K. Kataoka et al., JMS-Pure Appl. Chem. A31 (11), pp. 1759-1769, 1994).
In one study, using a human cancer cell line model, a new formulation containing biodegradable amphiphilic diblock copolymer, monomethoxy poly(ethylene glycol)-block-poly (D,L-lactide) (m PEG-PDLLA) and paclitaxel (Genexol(copyright)-PM) and Taxol(copyright) showed comparable in vitro cytotoxicity at the same concentrations. The polymeric micellar formulation of paclitaxel produced an increase in a maximum tolerated dose (MTD) as compared with that of Taxol(copyright) when administered i.p. in vivo. This formulation was said to have advantages over the commercially available injectable preparation of Taxol(copyright) in terms of low toxicity levels and increased paclitaxel dose (2 to 3-fold higher levels) (Kim S. C. et al., J. Controlled Release, v.72, pp. 191-202, 2001).
The advantages mentioned by the above authors are related to the slow release of paclitaxel from the micelles, due to a strong hydrophobic association between paclitaxel and the high molecular weight m PEG-PDLLA. At the same time, according to the authors, additional studies of a polymeric micellar formulation, comprising paclitaxel in unusually high doses will be required to fully characterize the nature of toxicities and especially the more distant consequences this kind of treatment.
The present inventors have taken a principally different approach. They have made available novel compounds, comprising the residues of naturally occurring substances only. These compounds, numbered I through VI, in themselves have low toxicity. A single dose i.p. toxicity study in rats was carried out in accordance with the OECD principles of Good Laboratory Practice. It was found that the compounds I-VI, at a dose level of 100 mg/kg body weight did not produce mortality. The minimal lethal dose is thus above 100 mg/kg body weight for these compounds (I-VI).
Considering xe2x80x9cchemotherapyxe2x80x9d in it widest meaning, i.e. the administration of chemical agents for the prevention, treatment or alleviation of a medical condition, a manifold of similar problems arise. It is important to optimise efficacy, e.g. the uptake and target-specificity of the compound, its distribution in the body and its clearance, simultaneously as minimising the possible side-effects, risks to medical staff etc. Also the cost of production, ease of preparation, modes of administration, stability during storage etc must be taken into consideration. In particular, it is desirable to be able to increase the solubility and bio-availability of poorly soluble pharmaceutical agents, increasing their efficacy and reducing their side-effects.
Considering xe2x80x9cchemotherapyxe2x80x9d in its more specific meaning, i.e. the use of chemical agents to destroy cancer cells, it remains an urgent task to make available new substances and formulations, which at least exhibit improved efficacy and less side-effects, but preferably also improved characteristics concerning solubility, safety, stability etc.
In particular, it is desirable to make available a new formulation of paclitaxel, exhibiting improved stability, improved efficacy and reduced side-effects, compared to presently available formulations. Further problems and the corresponding innovative solutions will be evident from the following description and claims.
DE 40 32 187 (Hermes Fabrik pharmazeutischer Prxc3xa4parate Franz Gradinger GmbH and Co., DE) discloses various N-Retinoyl-L-aminomercapto compounds and their physiologically acceptable salts. The compounds are suggested for use in the systemic and topical treatment of diseases of the mucous membranes. A closer study of the structural formulas reveals that structural elements, central for the compounds (I-VI) according to the present invention are absent or different in DE 40 32 187. Notably, the compounds of DE 40 32 187 contain a sulphur in oxidation state xe2x88x922 and xe2x88x921 respectively. The physiological function, as well as the physical and chemical properties of these sulphur containing compounds are determined by this oxidation state. There is also no indication that these compounds would influence the properties of paclitaxel or other water-insoluble or sparingly soluble substances.
Kalemkerian et al., Activity of fenretinide plus chemotherapeutic agents in small-cell lung cancer cell lines, Cancer Chemother Pharmacol, (1999) 43:145-150. This article describes a synthetic retinoid which is both a potent inducer of apoptosis in cancer cells, and which may have the capability of enhancing the activity of other cytotoxic agents. All combination studies were performed with a range of concentrations of each individual agent and both agents together at a fixed ratio corresponding to the ratio of the IC50 values of each agent alone as identified in preliminary experiments. The authors state that their study does not make it possible to say whether the experimental agents interacted in a mutually exclusive or mutually nonexclusive manner. The issues of solubilisation and storage of paclitaxel or other water-insoluble or sparingly soluble pharmaceuticals is not discussed.
Zhang et al., An investigation of the antitumour activity and biodistribution of polymeric micellar paclitaxel, Cancer Chemother Pharmacol, (1997) 40:81-86. In this study, the conventional Cremophor Paclitaxel formulation was compared to a polymeric micellar paclitaxel, administered by i.p. injection. A biodegradable amphiphilic diblock copolymer, monomethoxy poly (ethylene glycol) block-poly (D,L-lactide) [mPEG-PDLLA] was used. The micellar formulation showed very promising results. The advantages mentioned are however related to the slow release of paclitaxel from the micelles, due to strong hydrophobic association between paclitaxel and the high molecular weight mPEG-PDLLA. Further, the toxicity and the long-term consequences of this slow release mode of administration need to be further studied.
The present inventors have found that therapeutically active compounds can be dissolved in micelles of a compound which itself displays the desired therapeutic activity or an activity favourably interacting with or potentiating the desired activity, and which compounds exhibit low toxicity. The present invention thus makes available a group of new compounds, N-(all-trans-Retinoyl)-L-cysteic acid (I), N-(13-cis-Retinoyl)-L-cysteic acid (II), N-(all-trans-Retinoyl)-L-homocysteic acid (III), N-(13-cis-Retinoyl).L-homocysteic acid (IV), N-(all-trans-Retinoyl)-L-cysteinesulfinic acid (V), and N-(13-cis-Retinoyl)-L-cysteinesulfinic acid (VI), which exhibit therapeutic effects per se, and which in combination with known pharmaceuticals exhibit a synergistic effect. In combination with cytotoxic or cytostatic pharmaceuticals, said novel compounds introduce improved possibilities to combat cancer. Further, the present invention discloses a possibility of making water-soluble formulations of water-insoluble or sparsely soluble pharmaceuticals, such as paclitaxel, with enhanced pharmacological activity and improved storage and handling properties.
The terms xe2x80x9cpotentiationxe2x80x9d and xe2x80x9cpotentiatingxe2x80x9d are used to define an action by which the therapeutic effect of two or more compounds, given simultaneously or substantially simultaneously, is greater than the effect of said compounds given separately.
The term xe2x80x9csimultaneouslyxe2x80x9d should in this context be interpreted broadly, i.e. encompassing both situations where two or more compounds are given in admixture, and situations where the compounds are administered separately, either via the same or different routes of administration, at the same time or sequentially, provided that the compounds exert their therapeutic influence in the body at the same or practically the same time.
The term xe2x80x9ccritical micell concentrationxe2x80x9d or xe2x80x9cCMCxe2x80x9d is a measure of the concentration of a solution component, which represents a critical value above which increasing concentration of said component forces the formation of micelles.
The present inventors have surprisingly found that N-(all-trans-Retinoyl)-L-cysteic acid (I),N-(13-cis-Retinoyl)-L-cysteic acid (II), N-(all-trans-Retinoyl)-L-homocysteic acid (III), N-(13-cis-Retinoyl)-L-homocysteic acid (IV), N-(all-trans-Retinoyl)-L-cysteinesulfinic acid (V), and N-(13-cis-Retinoyl)-L-cysteinesulfinic acid (VI) are capable of increasing the solubility of sparsely soluble compounds, as well as potentiating their therapeutic efficacy.
These novel compounds according to the present invention are amides of all-trans-retinoic acid or 13-cis-retinoic acid with L-cysteic acid (3-sulfo-L-alanine), L-homocysteic acid, L-cysteinesulfinic acid. The structural formulas of these compounds are presented below: 
The molecules of these compounds simultaneously exhibit a hydrophilic and a hydrophobic part in water solutions. In the form of salts, these compounds are capable of forming micelles in aqueous solutions at concentrations equal to or higher than the critical micelle concentration (CMC).
The present invention makes available the use of the above compounds, or derivatives thereof, for the manufacture of a medicament. The present invention also makes available the use of the above compounds, or a derivative thereof, for the manufacture of a medicament for the treatment of cancer.
Further, the present invention makes available a pharmaceutical composition comprising an active substance in a therapeutically effective amount, and one of the above compounds (compounds I-VI), or a derivative thereof. In particular, the present invention makes available a pharmaceutical composition wherein the active substance is a cytotoxic compound, and the potentiating compound is one of the above compounds (compounds I-VI), or a derivative thereof. According to one embodiment of the invention, said active substance is paclitaxel.
Another embodiment of the present invention is a method for potentiating the efficacy of a pharmaceutically active substance, wherein said substance is prepared in micellar form with at least one of the above compounds (compounds I-VI), or a derivative thereof.
Another embodiment is a method for increasing the solubility of a pharmaceutically active substance, wherein said substance is prepared in micellar form with at least one of the above compounds (compounds I-VI), or a derivative thereof.
Yet another embodiment is a method for improving the bio-availability of a pharmaceutically active substance, wherein said substance is prepared in micellar form with at least one of the above compounds (compounds I-VI), or a derivative thereof.
The present invention also makes available a method for the treatment of cancer, wherein a cytotoxic substance is mixed with at least one of the compounds above (compounds I-VI), or a derivative thereof, and delivered to a patient. In particular, the invention concerns such a method, wherein the cytotoxic substance is paclitaxel.
The inventors have shown that the poorly soluble compound paclitaxel can be dissolved in micelles of N-(all-trans-Retinoyl)-L-cysteic acid (I), N-(13-cis-Retinoyl)-L-cysteic acid (II), N-(all-trans-Retinoyl)-L-homocysteic acid (III), N-(13-cis-Retinoyl)-L-homocysteic acid (IV), N-(all-trans-Retinoyl)-L-cysteinesulfinic acid (V), and N-(13-cis-Retinoyl)-L-cysteinesulfinic acid (VI), creating mixed micelles. In this way, an excellent solubility of paclitaxel in the form of mixed micelles in saline was achieved. Solutions of these compounds (compounds I-VI) in saline were prepared in a wide range of concentrations, and added in MEM with 5% fetal bovine serum (FBS) to cultures of human breast adenocarcinoma (the MDA-MB-231 cell line).
Tests evaluating the cytotoxicity of the inventive compounds in the concentration 40 nM have shown that better results are obtained in the range 0,005 mg/ml to 5,0 mg/ml of the compounds in saline (initial concentrations). A maximum cell growth inhibition close to 38% was observed at the initial concentration 1 mg/ml (in saline) before addition to the adenocarcinoma cultures (the MDA-MB-231 cell line).
Tests evaluating the cytotoxicity of the inventive compounds in the concentration range 10xe2x88x9211 M to 106 M in cultures of MDA-MB-231 cells have revealed the following dependence: An increase of the concentrations of the inventive compounds led to the enhancement of cell growth inhibition, achieving a value close to 42% at the concentration 10xe2x88x926 M.
The cytotoxicity of the formulation of paclitaxel/compound (I-VI), and compounds I through VI alone, was compared with paclitaxel and Taxol(copyright) in cultures of MDA-MB-231 cell line. In the case of paclitaxel and Taxol(copyright), the cell growth inhibition approached 46% at concentrations close to the IC50 concentration.
In particular at the same paclitaxel concentration, the formulation of paclitaxel and compound I or paclitaxel and compound II, both at a molar ratio of the components of 1:5, exhibited a surprisingly high cell growth inhibition of 70%. The extent of cell growth inhibition using the commercially available Taxol(copyright) (positive control) was 45%. Already the cytotoxic action of compounds I or II alone, at a concentration of 40 nM, was close to 40%. The formulations of paclitaxel and compound I (or compound II) display an increasing cell growth inhibition within the molar ratio range 1:3-1:5 (paclitaxel:compound I (or compound II)). When further increasing the ratio of the components to 1:10, the extent of the cell growth inhibition remains practically unchanged.
The inventive formulation of N-(all-trans-Retinoyl)-L-cysteic acid (I), N-(13-cis-Retinoyl)-L-cysteic acid (II), N-(all-trans-Retinoyl)-L-homocysteic acid (III), N-(13-cis-Retinoyl)-L-homocysteic acid (IV), N-(all-trans-Retinoyl)-L-cysteinesulfinic acid (V), N-(13-cis-Retinoyl)-L-cysteinesulfinic acid (VI) and paclitaxel is prepared as follows: Solutions of paclitaxel and any compound (I-VI) in ethanol (or other aliphatic alcohol) are first prepared in appropriate concentrations. Then aliquots of these solutions are mixed to form a mixed solution with the desired molar ratio paclitaxel:compound (I-VI). The obtained solution can be stored for at least three months at low temperatures, without noticeable change in the properties of compound (I-VI). Moreover, the formulation retains its cytotoxic effects during prolonged storage. Before use, the solution is evaporated in vacuo to yield a waxy solid which is dissolved in saline or other commonly used vehicle for intravenous infusion to a patient. Taxol(copyright) (Bristol-Myers Squibb Co.) is a formulation containing Paclitaxel (6 mg), ethanol (396 mg) and Cremophor(copyright) EL (527 mg). The present inventors have shown that the inventive compounds, N-(all-trans-Retinoyl)-L-cysteic acid (I), N-(13-cis-Retinoyl)-L-cysteic acid (II), N-(all-trans-Retinoyl)-L-homocysteic acid (III), N-(13-cis-Retinoyl)-L-homocysteic acid (IV), N-(all-trans-Retinoyl)-L-cysteinesulfinic acid (V), and N-(13-cis-Retinoyl)-L-cysteinesulfinic acid (VI) have excellent solubility in the commercially available Taxol(copyright) preparation. It is thus possible to easily improve the conventional paclitaxel formulation using the inventive compounds. An ethanol solution is prepared of one of N-(all-trans-Retinoyl)-L-cysteic acid (I), N-(13-cis-Retinoyl)-L-cysteic acid (11), N-(all-trans-Retinoyl)-L-homocysteic acid (III), N-(13-cis-Retinoyl)-L-homocysteic acid (IV), N-(all-trans-Retinoyl)-L-cysteinesulfinic acid (V), or N-(13-cis-Retinoyl)-L-cysteinesulfinic acid (VI). The obtained solution is evaporated in vacuo to give waxy solid, whereupon Taxol(copyright) is added, dissolving the waxy solid. The Taxol(copyright) emulsion forms a liquid system with the compounds (I-VI) even at a molar ratio of paclitaxel to compound (I-VI) of more than 1:20.
Tests for evaluating the cytotoxicity of an improved paclitaxel formulation (Taxol(copyright) plus compound I-VI) at the molar ratios of paclitaxel:compound I (through compound VI) from 1:1 to 1:20, were carried out in cultures of human breast adenocarcinoma (the MDA-MB-231 cell line). The results of these tests are similar to the results obtained for the formulation paclitaxel and compound I (through VI) in saline. The extent of cell growth inhibition for this improved paclitaxel formulation (Taxol(copyright) and compound I-VI) at the molar ratio 1:10 was increased by almost 50% (compared to that of Taxol(copyright) alone).
The present invention thus exemplifies the inventive concept, that sparsely soluble therapeutic agents can be made more soluble, and their therapeutic efficacy potentiated, by presenting twelve new therapeutic formulations comprising the anticancer drug paclitaxel:
paclitaxel and N-(all-trans-Retinoyl)-L-cysteic acid in saline,
paclitaxel and N-(13-cis-Retinoyl)-L-cysteic acid in saline,
paclitaxel and V-(all-trans-Retinoyl)-L-homocysteic acid in saline,
paclitaxel and N-(13-cis-Retinoyl)-L-homocysteic acid in saline,
paclitaxel and N-(all-trans-Retinoyl)-L-cysteinesulfinic acid in saline,
pacditaxel and N-(13-cis-Retinoyl)-L-cysteinesulfinic acid in saline,
Taxol(copyright) and N-(all-trans-Retinoyl)-L-cysteic acid,
Taxol(copyright) and N-(13-cis-Retinoyl)-L-cysteic acid,
Taxol(copyright) and N-(all-trans-Retinoyl)-L-homocysteic acid,
Taxol(copyright) and N-(13-cis-Retinoyl)-L-homocysteic acid,
Taxol(copyright) and N-(all-trans-Retinoyl)-L-cysteinesulfinic acid, and
Taxol(copyright) and N-(13-cis-Retinoyl)-L-cysteinesulfinic acid.
These formulations showed both good physical and chemical stability, which is believed to reduce the effects connected with paclitaxel precipitation upon dilution. This is also believed to solve the issues related to the stringent requirements regarding facilities and vessels for preparation and storage of conventional paclitaxel preparations.
Notably, the compounds (I-VI) have low toxicity, but display significant cell growth inhibition, the effect increasing in the appropriate concentration ranges.
The results obtained by the present inventors have laid a foundation for the development of a technique for large-scale synthesis of the compounds (I-VI), pharmaceutically useful salts thereof, and in particular Na-salts thereof. The synthesis of the compounds (I-VI) of the invention involves a direct acylation of the amino groups of L-cysteic acid, L-homocysteic, and L-cysteinesulfinic acid by mixed carbonic-carboxylic acid anhydride in water-organic medium, containing Na2CO3. The solubility of the sodium salts of the compunds (I-VI) in 2-propanol-water mixtures make it possible to separate insoluble contaminants (inorganic salts and starting amino acids). The pure compounds (I-VI) are then obtained by precipitation from their concentrated solutions in 2-propanol-water using a methanol-2-propanol mixture.
The above method of synthesis developed by the inventors makes it possible to produce sodium salts of the compounds (I-VI) in good yields. The method is simple and timesaving. The final products can be prepared in pure form, without the need of chromatography. The compounds (I-VI) in the form of sodium salts can be stored in a solution of 2-propanol-water 2:1 (v/v) or ethanol-water 2:1 (v/v) for at least six months at low temperatures without any noticeable change in their properties. In order to prepare the formulations of the inventive compounds (I-VI) with paclitaxel or Taxol(copyright), the sodium salts of these compounds are easily converted into the corresponding acidic forms, and dissolved in methanol.
Tests evaluating the cytotoxicity of the compounds I through VI, in the form of sodium salts in the concentration range 10xe2x88x9211 M to 10xe2x88x926 M, have been performed in cultures of MDA-MB-231 cells, and revealed the following dependence: an increase of the concentrations of the inventive compounds led to an enhancement of cell growth inhibition, achieving a value close to 50% for compounds I and II; a value close to 35% for compounds III and IV; and a value close to 30% for compounds V and VI.
Sodium salts of the compounds I through VI were converted into the corresponding acidic forms of the compounds and dissolved in methanol, in order to prepare the formulations paclitaxel/compound (I-VI). At the same paclitaxel concentration, the formulation of paclitaxel and compound I, or paclitaxel and compound II, exhibited a high cell growth inhibition close to 70% (close to 45% compared to paclitaxel alone as positive control); the formulation of paclitaxel and compound III or paclitaxel and compound IV exhibited a cell growth inhibition close to 60% (close to 30% compared to paclitaxel alone as positive control); the formulation of paclitaxel and compound V or paclitaxel and compound VI exhibited a cell growth inhibition close to 55% (close to 25% compared to paclitaxel as positive control). The molar ratio of paclitaxel:compound (I-VI) was 1:7.
Sodium salts of the compounds (I-VI) were converted into the corresponding acidic forms of the compounds and dissolved in Taxol(copyright) to prepare the formulations Taxol(copyright)/compound (I-VI).
The extent of cell growth inhibition for the formulation of Taxol(copyright)/compound I or Taxol(copyright)/compound II was close to 75% (close to 50% compared to Taxol(copyright) alone as positive control); for the formulation Taxol(copyright)/compound III or Taxol(copyright)/compound IV, the inhibition was close to 65% (close to 35% compared to Taxol(copyright) alone as positive control); for formulation Taxol(copyright)/compound V or Taxol(copyright)/compound VI, close to 60% (close to 30% compared to Taxol(copyright) alone as positive control). The molar ratio of paclitaxel:compound (I-VI) was 1:10.
The compounds (I-VI) surprisingly combine an ability for growth inhibition of tumour cells with the power to dissolve paclitaxel, creating mixed micelles in saline. The inventors further show that these compounds (I-VI) are able potentiate the efficacy of paclitaxel. The present inventors have also developed a method of production and produced a lyophilized composition of compound (I-VI)/paclitaxel in mixed micellar systems.
The optimisation of mixed-micellar systems of compounds (I-VI)/paclitaxel was performed using different molar ratios of paclitaxel:compound (I-VI) and vehicle. The mixed-micellar systems according to the invention did not cause precipitation of the drug upon dilution 100 times and more in water solutions.
Solutions of paclitaxel and the compound (I-VI) in methanol were mixed at different molar ratios paclitaxel:compound (I-VI) equal to 1:3, 1:5, and 1:7. After evaporation of the organic solvent under reduced pressure, the resulting dried film was dissolved by the addition of distilled water or 0.05 M sodium acetate buffer, pH 5.6 or 10% solution of ethanol or 0.15 M solution of NaCl or 0.05 M sodium acetate buffer, pH 5.6 in 10% solution of ethanol to obtain a mixed-micellar solution of compound (I-VI)/paclitaxel. These solutions were filtered through a 0.22 xcexcm sterile filter and stored at 4xc2x0 C. All of these prototype systems produced significant antitumor activity in vitro for three weeks. No precipitation or other gross changes were observed during storage.
The mixed micelles did not appear to be very stable in solution. The preparations of compound (I-VI)/paclitaxel in mixed-micellar systems at the molar ratio paclitaxel compound (I-VI) of 1:7 were freeze-dried (as water solution, W or solution in 0.05 M sodium acetate buffer, pH 5.6, SAB) and stored in powder form during 6 months at 4xc2x0 C. The preparations of compound (I-VI)/paclitaxel in mixed-micellar systems in dry form was shown to be stable for a sufficient period of time awaiting usage. There was no change in the concentration of the active ingredients at least during a 6-months storage at 4xc2x0 C.
Upon reconstitution with either distilled water, or 0.05 M sodium acetate buffer, pH 5.6, or 10% ethanol, or 0.15 M solution of NaCl, or 0.05 M sodium acetate buffer, pH 5.6 in 10% ethanol, a clear solution was obtained immediately.
The preparations of the dried compound (I-VI)/paclitaxel in mixed-micellar systems (OF) which were reconstituted with 0.05 M sodium acetate buffer (W/SAB) or the ones that were freeze-dried as the solution in 0.05 M sodium acetate buffer and reconstituted with water (SAB/W) exhibit the best cytotoxic action on MDA-MB-231 cell line. The cytotoxic action was similar to that of compound (I-VI)/paclitaxel formulations in saline (Table 1):
The inventive formulations compare favourably with Taxol(copyright), but are believed to remove or alleviate the adverse effects associated to Cremophor(copyright) EL. Tests in vitro show remarkable results, and there are substantial grounds to believe that the pharmacological activity in human patients is improved, compared to that of conventional paclitaxel formulations. Consequently, the present invention makes available a method for preparing a water-soluble formulation of paclitaxel, comprising the steps of dissolving paclitaxel in a first solvent, dissolving a compound (I-VI) in a second solvent, mixing the aliquots of the resulting solutions of paclitaxel and the said compound in a desired molar ratio, and evaporating the resulting mixture to dryness.
Further, the invention makes available a method for preparation a water-soluble improved formulation of Taxol(copyright), comprising the step of dissolving a compound (I-VI) in a solvent, evaporating the desired aliquot of the resulting solution to dryness and dissolving the residue in Taxol(copyright).
Further, the invention makes available a method for preparation a stable storage formulation of paclitaxel, comprising the steps of dissolving paclitaxel in a first solvent, dissolving a compound (I-VI) in a second solvent, mixing the aliquots of the resulting solutions of paclitaxel and the said compound in a desired molar ratio.
Further, the invention makes available a method for preparing the formulation of paclitaxel for administration to a patient, comprising the steps of dissolving paclitaxel in a first solvent, dissolving a compound (I-VI) in a second solvent, mixing the aliquots of the resulting solutions of paclitaxel and the said compound in a desired molar ratio, evaporating the resulting mixture to dryness forming a residue of paclitaxel and the said compound, dissolving the said residue in a aqueous solution, lyophilisation of the solution formed, followed by reconstitution of the lyophilised product using a vehicle suitable for administration to a patient.
The invention will be illustrated in closer detail in the following non-limiting examples.